Many genetic diseases are caused by the absence or mutation of the appropriate protein, for example as a result of deletions within the corresponding gene. One of the most common fatal genetic diseases in humans is cystic fibrosis (CF). Cystic fibrosis (CF), a spectrum of exocrine tissue dysfunction, which eventually leads to respiratory failure and death results from a mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene has now been to chromosome 7q31, and cloned. A 3 bp deletion, resulting in the loss of a phenylalanine residue at amino acid position 508, is present in approximately 70% of CF chromosomes, but is not seen on normal chromosomes. The other 30% of CF mutations are heterogeneous and include deletion, missense, and splice-site mutations. Transfection of even a single normal copy of the CFTR gene abolishes the CF secretory defect in CF cell lines, an observation which supports the feasibility of gene therapy for CF. These results demonstrate that expression of a wild-type CFTR transgene can exert a dominant positive effect in CF cells which concurrently express an endogenous mutant CFTR gene. Thus, expression of the wild-type CFTR transgene in the lungs of CF patients can correct the CF phenotype. However, to date, the inability to produce high level expression of transgenes in the lung by either aerosol or intravenous (iv) administration has precluded the use of gene therapy for the treatment of CF. Expression of a wild-type CFTR transgene in cells from CF patients corrects the chloride secretory defect, the primary biochemical lesion of CF. Chloride secretion is normalized in cells of CF patients despite the presence of the mutant CFTR protein, indicating that when wild-type and mutant CFTR proteins are coexpressed in cells, the wild-type CFTR is dominant.
To date, attempts to replace absent or mutated genes in human patients have relied on ex vivo techniques. Ex vivo techniques include, but are not limited to, transformation of cells in vitro with either naked DNA or DNA encapsulated in liposomes, followed by introduction into a suitable host organ ("ex vivo" gene therapy). The criteria for a suitable organ include that the target organ for implantation is the site of the relevant disease, the disease is easily accessible, that it can be manipulated in vitro, that it is susceptible to genetic modification methods and ideally, it should contain either non-replicating cells or cycling stem cells to perpetuate a genetic correction. It also should be possible to reimplant the genetically modified cells into the organism in a functional and stable form. A further requirement for ex vivo gene therapy, if for example a retroviral vector is used, is that the cells be pre-mitotic; post-mitotic cells are refractory to infection with retroviral vectors. There are several drawbacks to ex vivo therapy. For example, if only differentiated, replicating cells are infected, the newly introduced gene function will be lost as those cells mature and die. Ex vivo approaches also can be used to transfect only a limited number of cells and cannot be used to transfect cells which are not first removed from the body. Exemplary of a target organ which meets the criteria of in vivo gene transfer is mammalian bone marrow; mammalian lung is not a good candidate for ex vivo therapy.
Retroviruses, adenoviruses and liposomes have been used in animal model studies in attempts to increase the efficiency of gene transfer. Liposomes have been used effectively to introduce drugs, radiotherapeutic agents, enzymes, uses, transcription factors and other cellular effect into a variety of cultured cell lines and animals. In addition, successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed. Several strategies have been devised to increase the effectiveness of liposome-mediated drug delivery by targeting liposomes to specific tissues and specific cell types. However, while the basic methodology for using liposome-mediated vectors is well developed, the technique has not been perfected for liposome-based transfection vectors for in vivo gene therapy. In the studies published to date, injection of the vectors either intravenously, intratracheally or into specific tissues has resulted in low but demonstrable expression, but the expression has generally been limited to one tissue, typically either the tissue that was injected (for example muscle); liver or lung where iv injection has been used; or lung where intratracheal injection has been used, and less than 1% of all cells within these tissues were transfected.
In vivo expression of transgenes has been restricted to injection of transgenes directly into a specific tissue, such as direct intratracheal, intramuscular or intraarterial injection of naked DNA or of DNA-cationic liposome complexes, or to ex vivo transfection of host cells, with subsequent reinfusion. Currently available gene delivery strategies consistently have failed to produce a high level and/or generalized transgene expression in vivo. Expression of introduced genes, either complexed to cationic vectors or packaged in adenoviral vectors has been demonstrated in the lungs of rodents after intratracheal (IT) instillation. However, IT injection is invasive and produces a non-uniform distribution of the instilled material; it also is too invasive to be performed repeatedly in humans. For CF patients wherein the defect is a primary life-threatening defect in the lung, it would be of interest to develop a non-invasive delivery technique which also results in deeper penetration of exogenous nucleic acid constructs into the lung than do other methods, and can be used to deposit the CFTR gene constructs throughout the distal airways, as well as transfecting both airway epithelial cell and airway sub-mucosal cell types. Where other organs in the CF patient are affected due to the presence of mutant CFTR gene, techniques for transformation of a wide variety of tissues would be of interest, in order to alleviate extrapulmonary organ dysfunction in CF patients.
Relevant Literature
EP 91301819.8 (publication number 0 446 017 A1) discloses full length isolated DNAs encoding cystic fibrosis transmembrance conductance regulator (CFTR) protein and a variety of mutants thereof. Transient expression of CFTR in transformed cultured COS-7 cells is also disclosed. Rich et al., Nature (1990) 347:358-363 and Gregory et al., Nature (1990) 347:382-386 disclosed expression of the cystic fibrosis transmembrance conductance regulator in cultured HeLa cells using a vaccinia virus vector. Yoshimura et al. disclose expression of the CFTR gene in mouse lung after intracheal administration of a plasmid containing the gene, either as naked DNA or complexed to lipofectin.
Brigham et al., Am. J. Med. Sci. (1989) 22:278-281, describes the in vivo transfection of murine lungs with the CAT gene using a liposome vehicle. Transfection was accomplished by intravenous, intracheal or intraperitoneal injection. Both intravenous and intratracheal administration resulted in the expression of the CAT gene in the lungs. However, intraperitoneal administration did not. See, also Werthers, Clinical Research (1991) 39:(Abstract).
Canonico et al., Clin. Res. (1991) 39:219A describes the expression of the human .alpha.-1 antitrypsin gene, driven by the CMV promoter, in cultured bovine lung epithelial cells. The gene was added to cells in culture using cationic liposomes. The experimenters also detected the presence of .alpha.-1 antitrypsin in histological sections of the lung of New Zealand white rabbits following the intravenous delivery of gene constructs complexed to liposomes. Yoshimura et al. disclose expression of the human cystic fibrosis transmembrane conductance regulator gene in mouse lung after intratracheal plasmid-mediated gene transfer.
Multiple approaches for introducing functional new genetic material into cells, both in vitro and in vivo have been attempted (Friedmann (1989) Science, 244:1275-1280). These approaches include integration of the gene to be expressed into modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.: 5074S-5079S); integration into non-virus vectors (Rosenfeld, et al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434); or delivery of a transgene linked to a heterologous promoter-enhancer element via liposomes (Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; Nabel, et al. (1990) Science, 249:1285-1288; Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); coupled to ligand-specific, cation-based transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624) or the use of naked DNA expression vectors (Nabel et al. (1990), supra); Wolff et al. (1990) Science, 247:1465-1468). Direct injection of transgenes into tissue produces only localized expression (Rosenfeld (1992) supra); Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra). The Brigham et al. group (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) have reported in vivo transfection only of lungs of mice following either intravenous or intratracheal administration of a DNA liposome complex. An example of a review article of human gene therapy procedures is: Anderson, Science (1992) 256:808-813.
PCT/US90/01515 (Felgner et al.) is directed to methods for delivering a gene coding for a pharmaceutical or immunogenic polypeptide to the interior of a cell of a vertebrate in vivo. Expression of the transgenes is limited to the tissue of injection. PCT/US90/05993 (Brigham) is directed to a method for obtaining expression of a transgene in mammalian lung cells following either iv or intratracheal injection of an expression construct. PCT 89/02469 and PCT 90/06997 are directed to ex vivo gene therapy, which is limited to expressing a transgene in cells that can be taken out of the body such as lymphocytes. PCT 89/12109 is likewise directed to ex vivo gene therapy. PCT 90/12878 is directed to an enhancer which provides a high level of expression both in transformed cell lines and in transgenic mice using ex vivo transformation.
Debs et al. disclose pentamidine uptake in the lung by aerosolization and delivery in liposomes. Am Rev Respir Dis (1987) 135: 731-737. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991) 1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.